Use of heat-shock protein 27 for cardiovascular disease prevention and treatment

ABSTRACT

A method of preventing or treating cardiovascular disease is provided. The method comprises administering heat shock protein 27 (HSP27), a co-factor, variant or analogue thereof. The cardiovascular disease can include atherosclerosis. A pharmaceutical composition comprising HSP27 for use in the prevention or treatment of cardiovascular disease is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority from U.S.provisional patent application Ser. No. 60/955,210, filed Aug. 10, 2007,the entire contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to cardiovascular disease. Moreparticularly, the present invention relates to a method of preventing ortreating cardiovascular disease using heat shock protein 27.

BACKGROUND OF THE INVENTION

Heat shock proteins are involved in a wide variety of processes, bothphysiological and pathological. Heat shock protein 27 (HSP27) is amember of the small heat shock protein family, which comprises membersranging from 15 to 30 kDa in size and which may be phosphorylated oroligomerized under various conditions. HSP27 is principally described asan intracellular chaperone capable of binding and stabilizing the actincytoskeleton in response to stress. In addition, HSP27 can bindcytochrome c and prevent downstream caspase activation, making it apotent anti-apoptotic protein. More recently, it was discovered thatHSP27 can interact with estrogen receptor β and reduce transcriptionalsignaling through the estrogen response element¹. Althoughmulti-faceted, the functions described for HSP27 have been solelythought to be within the confines of the cell membrane; however,extracellular release of HSP27 may be regulated and may have importanteffects on steps leading to the development of atherosclerosis.

While the role of estrogens in atherosclerotic coronary artery diseasehas enjoyed intense scrutiny over the past two decades or more, recentreappraisals of clinical trials and new experimental data strongly arguefor a second look at the role of not only “hormone replacement therapy”(HRT), but also novel hormone manipulation strategies for theprevention/attenuation of atherosclerosis. There are several importantcaveats to clinical studies of HRT that help explain the absence of theexpected cardiovascular benefits of HRT in post-menopausal women²⁻⁴. Aprincipal deficiency of these studies is the late introduction of HRT towomen, who, for example, in a Women's Health study, were on average 7years post-menopausal before HRT was commenced^(5, 6). Many of thesewomen may have been susceptible to the potential ravages ofatherosclerosis while deficient of ovarian hormones in the initial yearspost-menopause. Hence, when HRT was introduced, it may have been toolate to derive an “atheroprotective” (or reversal of atherosclerosis)effect.

The biological effects of estrogen are mediated by at least two cellularreceptors: ER alpha (ERα) and ER beta (ERβ) that belong to the classicalsteroid hormone receptor superfamily. When activated, the receptorstranslocate to the nucleus and modulate transcriptional activity throughinteractions with estrogen response elements (EREs). These receptorsalso participate in signaling cascades at the cell membrane, suggestiveof a function entirely independent of gene regulation⁷. Structurally,ERα and ERβ are subdivided into several functional domains includingligand binding, DNA binding, and both ligand-independent (AF-1) andligand-dependent (AF-2) activation domains. While the two receptorsshare considerable structural similarities, they derive functionalspecificity via differential tissue expression patterns and regions ofstructural diversity (e.g. the A/B domain where there is only 30%sequence identity between ERβ and ERα)^(8, 9). Moreover, ER ligandcomplexes produce different effects in different cells due to variableexpression of co-regulatory proteins (e.g. co-activators andco-repressors). Indeed, in some instances the physiological andpathophysiological response to hormones may reside with theseco-regulatory molecules, rather than solely with the receptorsthemselves.

Approximately 300 nuclear receptor (NR) associated proteins are known,typically as a result of yeast two hybrid screens that employ the NR as“bait” (cf. review by Smith and O'Malley)¹⁰. In general, these proteinsdo not bind DNA directly, but instead facilitate the interaction ofhormone receptors with DNA and other structural proteins—ultimatelyserving to facilitate (activator) or hinder (repressor) the activationof transcription.

For a variety of reasons ERβ has emerged as a key receptor in the vesselwall. For example, the expression of ERβ mRNA is markedly up-regulatedafter vascular injury in male arteries^(11, 12). Moreover, in malearteries, ERβ is the predominant receptor expressed in the intima, mediaand adventitia, and its expression correlates with the degree ofcalcification—a marker of severe atherosclerosis. Therefore, ERβ appearsto play an important yet unidentified role in the progression ofatherosclerosis. Yeast two-hybrid analysis revealed that HSP27 is an ERβassociated protein. HSP27 attenuated ERβ transcriptional activitypreserved endothelial cell homeostasis, and normal volunteershad >3-fold higher serum levels than those patients with angiographicevidence of coronary artery disease.

It has been observed that HSP27 may be a potential biomarker foratherosclerosis, with expression of HSP27 diminishing with theprogression of disease^(1, 13, 14). Serum levels of HSP27 have beenshown to be attenuated in patients with atherosclerosis compared tohealthy individuals¹³. HSP27 may be involved in long term vessel wallhomeostasis that is then lost with the progression of atherosclerosis.Although the mechanisms by which HSP27 may be “atheroprotective” are notyet elucidated, many believe that analogous to its effects in othertissues (e.g. nerve, gastromucosal and myocardium) it protects thevessel wall from stressful stimuli and prevents apoptosis^(15, 16).While this may in part explain why HSP27 levels have been shown to beacutely increased in the serum following myocardial ischemia¹⁴, HSP27also appears to be involved in the long-term maintenance of vessel wallhomeostasis that unfortunately may be lost with the progression of CAD.

It is, therefore, desirable to provide a method for preventing ortreating cardiovascular disease using HSP27.

SUMMARY OF THE INVENTION

It is an object of the present invention to obviate or mitigate at leastone disadvantage of previous methods of preventing or treating disease,particularly cardiovascular disease.

In a first aspect, the present invention provides a method forpreventing or treating cardiovascular disease in a subject comprisingadministering to said subject heat shock protein 27 (HSP27), aco-factor, variant or analogue thereof. The cardiovascular disease canbe atherosclerosis, and more particularly selected from the groupconsisting of coronary atherosclerosis, peripheral vascular disease andneo-intimal formation, for example. Surprisingly, HSP27 has been foundto be atheroprotective.

HSP27 modulates cholesterol trafficking and mediators of inflammation.In particular, it has been surprisingly found that extracellular HSP27may provide some degree of sex-specific protection against thedevelopment of disease. This may be due in part to its ability to bindthe scavenger receptor-A and prevent uptake of atherogenic lipid (e.g.acytelated low-density lipoprotein or acLDL) as well as attenuateinflammation.

Administration of one or more types of estrogen (for example,17-β-estradiol), specific estrogen receptor modulators or relatedcompounds may contribute to an increase in HSP27 expression and, thus, apossible therapeutic effect in reducing atherosclerosis.

In another aspect, there is provided a kit for preventing or treatingcardiovascular disease, the kit comprising heat shock protein 27, avariant, a co-factor, or analogue thereof, and instructions for use inpreventing or treating cardiovascular disease.

The present invention also provides a pharmaceutical compositioncomprising heat shock protein 27, a co-factor, variant or analoguethereof. The composition may further comprise apharmaceutically-acceptable diluent or carrier. The pharmaceuticalcomposition is particularly suitable for treating or preventingcardiovascular disease as described herein.

In another aspect of the present invention, there is provided a methodfor preventing or treating cardiovascular disease in a subjectcomprising administering to said subject a therapeutically effectiveamount of an estrogen receptor agonist, antagonist, co-factor, oranalogue thereof, such that heat shock protein 27 expression ismodulated. A pharmaceutical composition comprising an estrogen receptoragonist, antagonist, co-factor, variant or analogue thereof, for use intreating or preventing cardiovascular disease, such that heat shockprotein 27 expression is modulated, is also contemplated.

In one embodiment, the estrogen receptor is ERβ; however estrogenreceptors may be involved. As one example, an estrogen receptor agonistis an estrogen, such as 17-β-estradiol.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1 shows HSP27 serum levels.

FIG. 2 shows the effects of HSP27 over-expression on atheroscleroticlesion size in mice after 4 weeks of high fat diet.

FIG. 3A shows HSP27 serum levels in response to an atherogenic diet.FIG. 3B shows regression analysis comparing HSP27 serum levels withaortic en face lesion area in female mice.

FIG. 4 illustrates secretion of HSP27 from macrophages in response toestrogen and acLDL. FIG. 4A shows macrophages treated with 100 nM17β-estradiol (E2) for 24 hours. FIG. 4B is a Western blot showingmacrophages treated in culture with 100 nM E2 for increasing times. FIG.4C is a Western blot which shows that macrophages were treated inculture with increasing concentrations of acLDL (0-100 μg/ml) FIG. 4D isa Western blot showing increased HSP27 secretion after treatment ofacLDL and/or E2. FIG. 4E is a Western blot following estrogen treatment.FIG. 4F is a Western blot following treatment with acLDL. FIG. 4G showsco-localization of HSP27 and the lysosome (LAMP1) after treatment withestrogen in macrophages. FIG. 4H shows J774 cells transfected withHSP27-ECFP (pseudo-coloured green) were treated with 100 μg/ml acLDL foreither 1 hour (middle panel) or 24 hours (bottom panel) in the presenceof Lysotracker red.

FIG. 5 shows extracellular HSP27 co-localizing with the scavengerreceptor A (SR-A) on the surface of macrophages. FIG. 5A shows theeffects of recombinant HSP27 (5 μg/ml) administration to macrophages for2 hours at 4° C. FIG. 5B shows macrophages from SR-A null mice incubatedwith recombinant HSP27. FIG. 5C illustrates HSP27-ECFP secreted inresponse to acLDL and applied to naïve macrophages.

FIG. 6A illustrates extracellular HSP27 and its effects on uptake ofacLDL by macrophages. FIG. 6B shows decreased foam cell content inperitoneal macrophages from HSP27^(o/e)/apoE−/− mice compared to WTapoE−/− mice (3% vs. 21% lipid-laden cells).

FIG. 7 shows the effects of extracellular HSP27 on the release ofcytokines involved in the inflammatory response: IL-1β (FIG. 7A) andIL-10 (FIG. 7B).

FIG. 8 illustrates the effects of HSP27 over-expression on macrophageadhesion and migration. In FIG. 8A, peritoneal macrophages harvestedfrom apoE^(−/−) HSP27^(o/e) and apoE^(−/−) mice were plated on type Icollagen. In FIG. 8B cell nuclei were stained and counted. FIG. 8C showsperitoneal macrophages as in FIG. 8A subject to CytoSelect™ migrationassay for 24 hours and quantified.

FIG. 9 shows a Western blot of HSP27 upon estrogenic stimulation of ERs.

FIG. 10 shows a Western blot of HSP27 after stimulation of Erβ.

FIG. 11 illustrates the localization of HSP27 and Erβ to membrane-boundvesicles.

FIG. 12 shows a Western blot of HSP27 protein expression in macrophages,endothelial cells and smooth muscle cells.

FIG. 13 shows images of dissected mice aortas and a timeline of diet andblood collection.

FIG. 14 shows relative HSP27 change in ovariectomized HSP27over-expressing and non-over-expressing mice after high fat diet.

FIG. 15 shows whole aortas from HSP27 over-expression apoE−/− mice, andthe quantification of aortic en face lesions.

FIG. 16A shows sections from the aortic sinus of HSP27 over-expressingand non-overexpressing apoE−/− mice, and FIG. 16B aortic sinus lesionarea, cholesterol cleft area and foam cell area.

FIG. 17 shows HSP27 serum levels in male and females.

FIG. 18 shows intima to media ratio (I/M ratio) in HSP27 over-expressing(HSP27o/e) and non-overexpressing (WT) mice subjected to femoral arterywire injury.

DETAILED DESCRIPTION

Generally, the present invention provides a method for preventing ortreating cardiovascular disease in a subject. More particularly, thepresent invention provides a method for preventing or treatingcardiovascular disease in a subject comprising administering to saidsubject a therapeutically effective amount of heat shock protein 27(HSP27), a co-factor, variant or analogue thereof. The cardiovasculardisease can be atherosclerosis, and more particularly coronaryatherosclerosis, but also other more peripheral forms of atherosclerosis(e.g., involving the carotid or other peripheral arteries, such as inperipheral vascular disease), or in neo-intimal formation. In oneembodiment, the subject is a female subject, such as a human femalesubject; however, this approach to the detection and treatment ofsubjects can also be applied to human male subjects.

Similarly, the present invention provides a pharmaceutical compositioncomprising heat shock protein 27, a co-factor, variant or analoguethereof, optionally together with a pharmaceutically-acceptable diluentor carrier. The present invention also contemplates a kit for preventingor treating cardiovascular disease, the kit comprising heat shockprotein 27, a variant, a co-factor, or analogue thereof, andinstructions for use in preventing or treating cardiovascular disease.The kit can be used for preventing or treating cardiovascular disease,particularly atherosclerosis, and more particularly coronaryatherosclerosis, but also other more peripheral forms of atherosclerosis(e.g., involving the carotid or other peripheral arteries).

In another aspect of the present invention, there is provided a methodfor preventing or treating cardiovascular disease in a subjectcomprising administering to said subject a therapeutically effectiveamount of an estrogen receptor agonist, antagonist, co-factor, oranalogue thereof, such that heat shock protein 27 expression ismodulated. A pharmaceutical composition comprising an estrogen receptoragonist, antagonist, co-factor, variant or analogue thereof, for use intreating or preventing cardiovascular disease, such that heat shockprotein 27 expression is modulated, is also contemplated.

As used in the present application, a “co-factor” can be any natural orartificial endogenous or exogenous molecule, such as a nucleotide,protein, chemical compound, adjuvant or the like, which a) binds to,associates with or interacts with an estrogen or HSP27, or an estrogenor HSP27 variant or analogue; b) binds to or assists with the binding ofan estrogen or HSP27 to a receptor, either intra- or extracellularly; orc) binds to, associates with or interacts with an estrogen receptorincluding, but not limited to, ERβ.

As used in the present application, “cardiovascular disease” refers toany disease associated with the heart, brain, or vasculature, or anyorgan or tissue in communication with the vasculature. Particularly,cardiovascular diseases of interest in the present invention are thosewhich are associated with the arterial vasculature including, but notlimited to, carotid, coronary and/or peripheral arteries (e.g.,“restenosis” after balloon angioplasty and/or stent implantation, andtransplant vasculopathy).

As used in the present application, a “variant” can include a modifiedmolecule, such as a nucleotide or protein. Suitable variants can includemolecules which have been methylated, for example.

As used in the present application, an “analogue” is a molecule, such asan HSP27 nucleotide or protein sequence, which has been modified byinsertion, deletion or substitution of one or more nucleotide or aminoacid residues, such that the function of the molecule is maintained.

As used herein, “modulation” can refer to any induced modification on amolecular pathway such as, but not restricted to, upregulation ordownregulation of gene expression, protein translation, cell signallingor other process.

EXAMPLES Serum Collection

Plasma was collected from healthy controls (n=9) and patients withangiographic evidence of coronary artery disease (n=25) in accordancewith the University of Ottawa Heart Institute Research Ethics Board.There were 21 males and 13 females with a mean age of 65±3 years, and61±8 years, respectively. For mice, serum samples were collected atbaseline (before the commencement of high-fat diet), and after 2 and 4weeks of high-fat diet. Plasma and serum was stored at −80° C. untilfurther use.

Enzyme Linked Immunosorbent Assay

Plasma levels of HSP27 were measured using an ELISA specific to humanHSP27 (Calbiochem, San Diego, Calif.). A total of 50 μl of human plasmawas assayed from both healthy controls and patients with CAD accordingto the manufacturer's protocol. A standard curve of known amounts ofHSP27 was constructed with each assay. For mouse samples, serum wasdiluted 1:10 in dilution buffer, and assayed according to themanufacturer's protocol. This assay was found to have nocross-reactivity with mouse HSP25.

Mouse Model

Mice over-expressing human HSP27 (HSP27^(o/e)) on a C57BL10/CBAbackground were provided by Imperial College London, and were maintainedby continuously backcrossing with C57BL10/CBA mice. HSP27^(o/e) femaleswere crossed with apoE^(−/−) males to generate apoE^(+/−)HSP27^(o/e)mice, which were crossed again to apoE^(−/−) mice to generate apoE^(−/−)HSP27^(o/e) (n=6 males and n=9 females) and apoE^(−/−) (n=6 males andn=9 females) littermates. All mice were tail-clipped and genotyped usingpreviously described protocols¹⁷ Mice were fed a normal chow diet until6 weeks of age, wherein they received high-fat diet (1.25% cholesterol,15.8% fat; Harlan Teklad, Madison, Wis.) for 4 weeks. Scavenger receptorknock out mice (SR-A KO) were used for collection of peritonealmacrophages at 12 weeks of age.

Cell Culture

Mouse peritoneal macrophages were obtained from peritoneal lavage with 9ml sterile PBS. Cells were centrifuged and washed twice with PBS andresuspended in culture medium before counting and plating in 24-wellplates. Peritoneal macrophages and J774 mouse macrophages weremaintained in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen,Burlington, ON) supplemented with 10% fetal bovine serum (FBS, Wisent,Saint-Jean-Baptiste de Rouville, QC), penicillin-streptomycin(Invitrogen), and fungizone (Invitrogen). acLDL was obtained fromIntracel (Frederick, Md.), and Dil-labeled acLDL was obtained fromInvitrogen. Fucoidan was obtained from Sigma-Aldrich (Oakville, ON).Lysotracker® Red was obtained from Invitrogen. Recombinant HSP27 waspurchased from Stressgen (Ann Arbour, Mich.).

Transfections

J774 cells were transfected using Fugene HD transfection reagent (Roche,Laval, QC) as per the manufacturer's instructions. Cells were grown to90% confluence on coverslips and transfected at a ratio of 2:3(DNA:reagent) with HSP27-ECFP (pECFP-C1 was obtained from Clontech) for24 hours before any treatment was initiated.

Western Blotting

Cell lysates were obtained using a RIPA buffer [1% NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS, Complete™ inhibitor in PBS]. Cells were washedtwice with PBS, lysed in RIPA buffer, collected using a cell scraper,and placed on ice for 45 minutes. Cell debris was collected bycentrifugation at 15,000×g for 20 minutes, and the supernatantcontaining the cellular protein was isolated. Protein was quantifiedusing Bradford reagent (Sigma). For western blotting, 50 μg of wholecell protein was loaded onto a 10% SDS-PAGE gel and separated at 120Vusing gel electrophoresis. Protein was then transferred to a PVDFmembrane (BioRad, Hercules, Calif.) for 2 hours at 60V. Membranes werethen subjected to western blotting using the following antibodies:anti-HSP27 (Santa Cruz, 1:200); anti-SR-A (Chemicon, 1:2000); anti-IL-1β(R&D, 1:500); anti-IL-10 (Santa Cruz, 1:200).

Immunolabeling

J774 cells treated with recombinant HSP27 (5 μg/ml) with or withoutfucoidan (10 μg/ml) were fixed with BD Cytofix (BD Biosciences,Mississauga, ON) and blocked with 2% bovine serum albumin (BSA) for 2hours. Antibodies against human HSP27 (Chemicon, 1:200) and scavengerreceptor A (Serotec, 1:200) were incubated overnight at 4° C.Visualization of substrates was done using secondary antibodiesconjugated to fluoroscein (for HSP27) and Texas Red (for SR-A). Negativecontrols included incubation with control IgG and secondary antibodyalone.

Cross-Linking and Immunoprecipitations

J774 cells were transfected with HSP27-ECFP as described above, andtreated with 100 μg/ml acLDL for 24 hours. Conditioned media from thesecells was applied to naïve (untransfected) macrophages for 2 h at 4° C.to allow HSP27 to bind the cell surface. Cross-linking was performedusing 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP, Pierce,Rockford, Ill.) as per the manufacturer's instructions. Cells wereharvested in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl,10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mMPMSF, 0.5% NP-40 in PBS) and 100 μg of total protein wasimmunoprecipitated with 5 μg of anti-ECFP antibodies (A.V. peptide, BDBiosciences) overnight at 4° C. with shaking. Protein-G-agarose wasadded for 4 hours, following which cells were centrifuged at 14,000×gand washed four times in IP buffer. Finally, IP pellets were resuspendedin SDS-PAGE loading buffer with or without β-mercaptoethanol to reversethe cross-linking (reduced and non-reduced, respectively) and subjectedto western blotting.

Confocal Microscopy

Cells were grown on cover slips, and transfected as described above.Cells were fixed with BD Cytofix as directed by the manufacturer andmounted with Dako Fluorescent mounting media (Dako Cytomation,Mississauga, ON). Coverslips were visualized with an Olympus FluoViewFV1000 confocal microscope (Olympus America Inc, Center Valley, Pa.) at100× magnification, using sequential scanning to reduce any potentialnon-specific excitation of the different fluorophores.

Fluorescent acLDL Uptake

Cells were treated with Dil-acLDL (5 μg/ml) for 4 hours at 37° C., inthe presence of recombinant HSP27 (5 μg/ml) and/or fucoidan (10 μg/ml).Cells were harvested by gentle cell scraping in 100 μl of PBS. Eachsample was analyzed for acLDL fluorescence on a BMG PolarStar platereader (excitation: 510 nm, emission: 570 nm) and normalized to totalcell number using the Vi-Cell cell counter (Beckman Coulter,Mississauga, ON).

Cell Adhesion Assay

Peritoneal macrophages were harvested from apoE^(−/−) mice andapoE^(−/−) HSP27^(o/e) mice as described above. Cells (1.6×10⁵) wereplated on type I collagen-coated 35 mm-dishes in 10% FBS DMEM. After 2hours of incubation at 37° C., cells were washed with PBS and fixed with4% paraformaldehyde in PBS for 1 hr at 4° C. Cell nuclei were stainedwith Hoechst 33258 for 10 min at RT. Photos were taken underfluorescence microscopy at 10× magnification, and the number of attachedcells was manually counted per high power field (HPF).

Cell Migration Assay

Peritoneal macrophages were harvested from apoE^(−/−) mice andapoE^(−/−) HSP27^(o/e) mice as described above. Cells (5.0×10⁴) wereplated in the top chamber of a CytoSelect™ 96-Well Cell Migration Assay(Cell Biolabs, San Diego, Calif.) in 0.1% FBS-DMEM. The bottom chambercontained 10% FBS-DMEM as a chemoattractant. The migration chamber wasincubated at 37° C. overnight, and migrated cells were quantified usinga fluorescent dye (as per the manufacturer's instructions) and the BMGPolarStar plate reader (excitation: 488 nm, emission: 520 nm).

Statistical Analysis

All data represent mean±SEM. Statistical significance was defined by pvalue <0.05 and is denoted by an asterisk (*).

Example 1 Expression of HSP27 In Vivo

It has been suggested that HSP27 can serve as a biomarker for bothcarotid atherosclerosis and acute coronary syndrome¹⁴. As shown in FIG.1, patients suffering from stable coronary disease (as demonstratedangiographically by coronary stenosis greater than 50%) demonstrate a3-fold reduction in circulating HSP27 levels in the serum when comparedto healthy, age-matched controls (828±195 vs. 272±38 pg/ml; p≦0.05). Anegative correlation coefficient of −0.90 was observed. Serum HSP27levels from healthy controls are in the left column and patients withstable CAD are in the right column (*p=0.004). Thus, HSP27 expression inthe serum appears to be reduced in stable CAD. Patients with stable CADalso had reduced HSP27 serum levels when compared to those sufferingfrom acute coronary syndrome (ACS).

Example 2 Mouse Model of Atherosclerosis

Using a mouse model of inflammatory atherosclerosis, the effects ofover-expressing human HSP27 (HSP27^(o/e)) were examined in thedevelopment of disease. ApoE^(−/−) HSP27^(o/e) and apoE^(−/−) mice wereplaced on a high-cholesterol diet for 4 weeks and euthanized at 10-weeksof age. There was no difference in mean bodyweight or length betweenHSP27Tg and WT mice, nor where there any differences between total serumcholesterol levels between these mice. FIGS. 2A to 2G show that thepercentage aortic lesion area, measured by quantitative histomorphologyof en face specimens, was reduced by 41% in the female HSP27Tg/apoE−/−vs. WT/apoE−/− mice (p<0.001). Total aortic en face atheroscleroticlesion area was analyzed in mice over-expressing human HSP27 (apoE^(−/−)HSP27^(o/e)) (FIGS. 2A, 2C, 2E) and compared to their apoE^(−/−)littermates (FIGS. 2B, 2D, 2F). Quantification of lesion area/totalaortic area (FIG. 2G) demonstrates there was a 35% reduction in lesionburden in apoE^(−/−) HSP27^(o/e) female mice compared to apoE^(−/−)(*p<0.001). Interestingly, there was no difference observed between maleapoE^(−/−)HSP27^(o/e) and WT/apoE−/− mice, suggestive of a role ofovarian hormones in the atheroprotective effects of HSP27. The serum ofthese mice was examined for circulating HSP27 levels using an ELISA.

FIG. 3A shows that in all mice fed a normal chow diet, there was littleto no detectable HSP27 in the serum. However, following 4 weeks of ahigh-fat diet, female HSP27^(o/e)/apoE−/− mice had 10-fold highercirculating levels of HSP27 compared to male mice (p≦0.05). These werethe same female mice that were offered a 41% relative protection fromlesion development compared to their wild-type littermates—an effectthat was not observed in the male mice. FIG. 3B shows that whencirculating HSP27 levels were compared to total en face lesion area,there was a significant negative correlation between serum HSP27 levelsand atherosclerotic lesion area (r²=−0.90; p<0.001). Thus, femaleovarian hormones may be involved in atheroprotective effects of HSP27.

Example 3 HSP27 Secretion In Vitro in Response to Estrogen and acLDL

As serum HSP27 levels inversely correlate with aortic lesion area inmice fed a cholesterol-enriched diet, and is 10-fold higher in femalesthan in males, we determined in vitro whether HSP27 is released onstimulation with estradiol (E2) or atherogenic acetylated low-densitylipoprotein (acLDL). Human macrophages (U937) were plated in replicatesat a density of 1_(—)106 per well and treated with estradiol or acLDL.Conditioned media was collected and before analysis for secreted HSP27,overall cell viability was measured using an LDH-release assay andrevealed no difference in cell viability or membrane integrity betweenany of the treatments (data not shown). Treatment of macrophages withestrogen (E2) for 24 hours caused a dose-dependent increase in HSP27release into the media compared to controls (FIG. 4A). Estrogen-inducedHSP27 release also increased over time, with maximum secretion after 24hours (FIG. 4B). Macrophages were subjected to increasing concentrationsof acLDL (1 to 100 μg/mL) for 24 hours. HSP27 protein was detected byWestern blot in conditioned media from cells treated with 100 μg/mLacLDL (FIG. 4C). The addition of acLDL to the media containing estrogencaused a further increase in HSP27 secretion when compared to estrogenor acLDL treatment alone, indicating that these 2 mechanisms ofsecretion may act synergistically (FIG. 4D). On treatment with estrogen,examination of intracellular protein levels revealed that HSP27 proteinlevels increased slightly in response to estrogen treatment, suggestiveof an intracellular pool of HSP27 that is secreted without necessitatingde novo protein synthesis (FIG. 4E).

On treatment with acLDL, there was a concomitant dose-dependent increasein intracellular HSP27 expression, then an apparent decreasecorresponding to increased HSP27 protein release into the media (FIG.4F). To examine the pathway of HSP27 secretion, we used 2 independentexperiments: the first used human U937 macrophages treated with E2 andimmunolabeled for HSP27 and a marker of the lysosomal membrane (LAMP1);second, mouse J774 macrophages were transfected with a fluorescentlytagged HSP27 (HSP27-ECFP), and treated with acLDL in the presence ofLysotracker, which labels acidic organelles (ie, lysosomes) in livecells. Using both methods, we localized intracellular HSP27 inmacrophage lysosomes after treatment with E2 or acLDL. Specifically,human macrophages treated with 100 nmol/L E2 overnight displayedcolocalization of HSP27 (red) and LAMP1 (green; FIG. 4G). Similarly,mouse macrophages transfected with fluorescently-tagged HSP27 (green)treated with 100 μg/mL acLDL and incubated in the presence ofLysotracker (red) displayed HSP27 colocalization within lysosomes aftertreatment for 1 hour and 24 hours (merged image, FIG. 4H). Withoutestrogen treatment, HSP27 showed minimal colocalization with thelysosome (FIGS. 4G and 4H, bottom row). Cells transfected with emptyECFP alone with or without acLDL treatment did not show colocalizationwith the lysosome, indicating that HSP27-ECFP was not simply degradedand targeted to the lysosome (data not shown). These results indicatethat HSP27 is found within secretory lysosomal-like vesicles inmacrophages under conditions which stimulate its secretion (eg, ontreatment with E2 or acLDL).

Example 4 Extracellular HSP27 Binding of the Scavenger Receptor-A InVitro Results in Decreased acLDL Uptake and Inflammation

As HSP27 is secreted not only in vitro by atherogenic lipids, but invivo in response to high fat diet, extracellular HSP27 binding of areceptor on the surface of cells to exert its potential atheroprotectiveeffects was examined. As is known in the prior art, members of the heatshock protein family bind a variety of cell surface receptors, includingtoll-like receptors and scavenger receptors¹⁸⁻²¹. Due to the observationthat higher levels of HSP27 correlate with lower atherosclerotic lesionburden, extracellular HSP27 was examined for possible binding of theSR-A receptor—an important receptor for the uptake of atherogenic lipidsand the progression of atherosclerosis^(22, 23).

FIG. 5A shows immunolabeling studies which revealed that recombinantHSP27 is capable of binding the surface of macrophages, and co-localizeswith SR-A. Immunolabeling of HSP27 (green) and SR-A (red) was visualizedunder confocal microscopy (top panel). Cells were also treated with anSR-A specific competitive ligand (fucoidan, 10 μg/ml) beforeadministration of HSP27 (bottom panel). Co-localization is seen as ayellow colour. In the presence of fucoidan, a specific competitor forSR-A, HSP27 binding to SR-A was reduced, indicating that thisinteraction is specific. FIG. 5B shows that, in mouse macrophages fromSR-A null mice, HSP27 binding to the cell surface was completelyabolished. Macrophages from SR-A null mice were harvested and incubatedwith recombinant HSP27 (5 μg/ml) for 2 hours at 4° C. Immunolabeling wasperformed as described above.

Endogenous HSP27 secreted in response to acLDL was examined to determineif it was also capable of binding the SR-A. Conditioned media frommacrophage whole cell lysates were transfected with HSP27-ECFP andtreated with 100 μg/ml acLDL was applied to naïve, untreated macrophagesat 4° C. for 2 hours to allow HSP27-ECFP to bind the cell surface. Cellswere then treated with DTSSP, a reversible, membrane-impermeablecross-linking agent, to cross-link HSP27 to the cell surface.Immunoprecipitation was carried out using antibodies to the fluorescenttag (anti-ECFP) and cross-linked proteins were either reduced (toreverse cross-linking) or non-reduced (to maintain it), separated on anSDS-PAGE gel and subjected to immunoblotting.

FIG. 5C shows that using antibodies to SR-A, HSP27-ECFP secreted fromcells treated with acLDL binds the scavenger receptor-A, under variousconcentrations of DTSSP. HSP27-ECFP secreted in response to acLDL wasapplied to naïve macrophages at 4° C. for 2 hours. Cells werecrosslinked with increasing concentrations of DTSSP (0.1-2 mM) andimmunoprecipitated using an antibody to ECFP.

Whole cell lysate from macrophages was also probed showing the detectionof a ˜50 kDa band corresponding to SR-A in these cells. This dataconfirms that indeed HSP27 binds specifically to SR-A both as anextracellular recombinant protein, and as a protein secreted in responseto acLDL.

Foam cell formation is a hallmark process in the development ofatherosclerosis, and prevention of lipid uptake by macrophages may serveto reduce overall lesion development and inflammation. If HSP27 can bindthe SR-A in vitro, and there is an atheroprotective effect of HSP27 invivo, HSP27 may prevent the SR-A from taking up atherogenic lipids suchas acLDL and becoming foam cells. In support of this, mouse macrophageswere cultured in vitro in the presence of fluorescently labeled acLDLfor 6 hours, and acLDL uptake was measured using a fluorometer andnormalized to total cell number.

FIG. 6A shows that when extracellular HSP27 was added to the culturemedia, there was a 41% reduction in acLDL uptake by macrophages(p≦0.05). Extracellular HSP27 inhibits uptake of acLDL by macrophagesvia the scavenger receptor A. Fluorescently labeled acLDL (5 ug/ml) wasgiven to macrophages in the presence (right columns) or absence (leftcolumns) of recombinant HSP27 (5 μg/ml). Cells were also treated with anSR-A specific competitor (fucoidan, 10 ug/ml) before administration ofacLDL or HSP27. Fluorescent uptake was measured and normalized to totalcell number. Fucoidan, a specific SR-A competitive ligand, was capableof inhibiting this process independently of the presence of HSP27,indicating that this reduction in acLDL uptake by HSP27 is likely viathe SR-A. FIG. 6B shows decreased foam cell content in peritonealmacrophages from HSP27^(o/e)/apoE−/− mice compared to WT apoE−/− mice(3% vs. 21% lipid-laden cells).

FIG. 7 shows that extracellular HSP27 results in release of cytokinesinvolved in the inflammatory response. Fluorescently labeled acLDL (5ug/ml) was given to macrophages in the presence or absence ofrecombinant HSP27 (5 μg/ml). Conditioned media was collected after 30minutes and 4 hours and subjected to western blotting for IL-1β (FIG.7A) and IL-10 (FIG. 7B). Addition of HSP27 caused a decrease in theacLDL-induced secretion of IL-1β into the extracellular space.

Moreover, this reduction in acLDL uptake by HSP27 resulted in aconsiderable decrease in the acLDL-induced release of IL-1β bymacrophages, a potent pro-inflammatory cytokine (FIG. 7A). ExtracellularHSP27 also increased the released of the anti-inflammatory cytokineIL-10 (FIG. 7B). Thus, it appears that extracellular HSP27 is primarilyan anti-inflammatory signaling protein. This is consistent with previousstudies in monocytes.

In summary, in vitro studies of mouse macrophages in culture subjectedto increasing concentrations of atherogenic lipid (acLDL) revealed thatHSP27 is secreted by macrophages (lysosomal) in response to acLDL. Theresults suggest that HSP27 is secreted by macrophages in vitro inresponse to acLDL. HSP27 was observed to bind to the surface ofmacrophages and interact specifically with SR-A; in SR-A null mice,HSP27 binding to the cell surface was completely abolished. It wasconfirmed that HSP27 binds specifically to SR-A both as extracellularrecombinant protein (HSP27-ECFP), and as a protein secreted in responseto acLDL. Thus, the results suggest that extracellular HSP27 binds thescavenger receptor-A. Further, it was found that the reduction in acLDLuptake by HSP27 is via the SR-A on road to foam cell development. Thereduction in acLDL uptake by HSP27 resulted in a considerable decreasein acLDL-induced release of IL-1β by macrophages, a potentpro-inflammatory cytokine, and an increase in IL-10, andanti-inflammatory cytokine. Thus, the results suggest that intracellularHSP27 prevents acLDL uptake by macrophages.

Example 5 HSP27 Over-Expression Reduces Cell Adhesion and Migration

To further investigate how HSP27 might be protective against thedevelopment of atherosclerosis, peritoneal macrophages were harvestedfrom HSP27^(o/e)/apoE−/− and WT apoE−/− mice after a high fat diet. FIG.8 shows that HSP27 over-expression results in decreased macrophageadhesion and migration. Peritoneal macrophages harvested fromapoE^(−/−)HSP27^(o/e) and apoE^(−/−) mice were plated on type Icollagen. After 2 hrs incubation, the cells were washed and fixed. Cellnuclei were stained with Hoechst 33258 and the number of cells per highpower field (HPF) was manually counted. Peritoneal macrophages as inFIG. 8A were subject to CytoSelect™ migration assay for 24 hours, andquantified as total number of cells migrated towards 10% FBS. Cells wereplated in culture on a collagen matrix, and allowed to adhere for 2hours. As shown in FIGS. 8A and 8B, there was a 53% reduction in celladhesion in HSP27^(o/e)/apoE−/− macrophages compared to WT apoE−/−(p≦0.001). Macrophages were also placed in a transwell migration chamberin serum-free media, and allowed to migrate towards 10% FBS overnight.FIG. 8C shows a 42% reduction in cell migration in HSP27^(o/e)/apoE−/−macrophages compared to WT (p≦0.01). Combined, these results indicatethat macrophages from HSP27 over-expressing mice have a reduced abilityto adhere and migrate, suggesting that in vivo these cells are lesslikely to incorporate into vascular lesions and exacerbate disease

Thus, In vitro adhesion and migration experiments using peritonealmacrophages from HSP27 and WT mice after a high diet revealed thatmacrophages from HSP27 over-expressing mice have a reduced ability toadhere and migrate, suggesting that in vivo, these cells are less likelyto incorporate into vascular lesions and exacerbate disease. The resultssuggest that HSP27 over-expression reduces cell adhesion and migration.

The methodologies used are described herein.

Example 7 Release of HSP27 and Interaction with Estrogen Receptors (ERs)

FIG. 9 shows that HSP27 secretion may be dependent upon estrogenicstimulation of ERs. Human macrophages were treated with increasingconcentrations of estrogen (E2; 0-100 nM) with or without increasingconcentrations of the ER-antagonist ICI 182,780 (0-100 nM). HSP27 wasdetected in the extracellular space by Western blot. The bottom panel ofFIG. 10 shows a quantification of the bands.

In FIG. 10, HSP27 secretion was shown to be enhanced upon stimulation ofERβ. Human macrophages were treated with estrogen (E2; 100 nM), anEra-specific agonist (PPT; 10 nM), or an Erβ-specific agonist (DPN, 1nM) for either 4 h or 24 h. HSP27 was detected in the extracellularspace by Western blot. The bottom panel of FIG. 10 shows thequantification of bands.

FIG. 11 shows an example of HSP27 and Erβ localizing to membrane-boundvesicles. Human macrophages were treated with E2-BSA (cell-impermeableE2) labeled with FITC, and immunolabeled for HSP27 and ERβ. The mergedimage shows HSP27 and Erβ colocalizing with vesicles at the membranecontaining E2-BSA.

FIG. 12 shows the release of HSP27 in a cell-type specific manner. Humanmacrophages, aortic endothelial cells and aortic smooth muscle cellswere treated with estrogen (E2; 100 nM), an Era-specific agonist (PPT;10 nM), or an Erβ-specific agonist (DPN, 1 nM) for 24 h. HSP27 wasdetected in the extracellular space by Western blot. Erβ stimulationappeared to cause an increase in HSP27 secretion from endothelial cellsbut not smooth muscle cells.

In FIG. 13, HSP27 over-expressing mice (HSP27o/eapoE−/−) ornon-overexpressing mice (apoE−/−) were ovariectomized at 6 weeks of ageand 2 weeks later placed on high fat diet. Mice were sacrificed at 12weeks of age. Aortas were dissected and reveal no difference in lesionarea in HSP27 over-expressing versus non-overexpressing mice. Thisappears to support previous data showing a 35% reduction in lesion areain female mice with the presence of endogenous estrogen (i.e., with theovaries intact). These results suggest that HSP27-mediatedatheroprotection requires endogenous estrogen in vivo.

FIG. 14 shows that HSP27 serum levels remain low in ovariectomized mice.Serum was collected from HSP27 over-expressing mice (HSP27o/eapoE−/−) ornon-over-expressing mice (apoE−/−) that were ovariectomized at 6 weeksof age and 2 weeks later placed on high fat diet. HSP27 ELISA analysisof the serum samples demonstrated that with the exception of 1 mouse(#1125) mice showed virtually no change in their serum HSP27 levels,indicating that high levels of release of this protein appear to beestrogen dependent.

Example 8 Gender-Related Protective Effects of HSP27 in Atherosclerosis

FIG. 15(A) shows whole aortas from HSP27 over-expressing apoE−/−mice ornon-overexpressing apoE−/− littermates after long term (12 week)atherogenic diet. FIG. 16(B) shows a quantification of aortic en facelesions expressed as lesion area as a % of total aortic area.

In FIG. 16(A), the aortic sinus of HSP27 over-expressing apoE−/−mice andnon-overexpressing apoE−/− littermates was sectioned and stained forMasson's trichromestain (first 2 columns) and anti-Mac1 (last 2 columns)which labels macrophages, after long term (12 weeks) atherogenic diet.FIG. 16(B) shows aortic sinus lesion area (first graph) cholesterolcleft area (second graph) and foam cell area (third graph)quantification. This appears to suggest that HSP27 over-expressionreduces foam cell formation and cholesterol cleft content inatherosclerotic lesions.

FIG. 17 shows HSP27 serum levels measured using an ELISA kit beforeatherogenic diet, 4 weeks post-atherogenic diet and 12 weekspost-atherogenic diet. The results show that HSP27 serum levels increasewith the duration of atherogenic diet in males and females.

Example 9 Effect of Chronic Over-Expression of Heat Shock Protein 27Reduces the Foam Cell Content of Atherosclerotic Lesions in Both Maleand Female ApoE^(−/−) Mice

The goals of this study were i) to explore if chronic over-expression ofHSP27 can provide persistent atheroprotection in females; and ii) ifmale mice can ultimately benefit from the effects of HSP27 on foam cellformation.

Materials and methods: For a detailed account of the Materials andMethods used in this study, please see the Supplemental Materialsavailable online at http://circres.ahajournals.org

Reduction in Atherosclerotic Lesion Area in HSP27 Over-expressing Mice:Male and female HSP27 over-expressing mice crossed to an apoE^(−/−)background (apoE^(−/−)HSP27^(o/e)) and non-overexpressing apoE^(−/−)littermates (apoE^(−/−)) were placed on a high-fat diet for 12 weeks.Female apoE^(−/−)HSP27^(o/e) mice had a 35% reduction in aortic en facelesion area compared to apoE^(−/−) females (p≦0.05; FIG. 15B). Aorticsinus lesion area was also reduced by 30% in these femaleapoE^(−/−)HSP27^(o/e) mice (p≦0.05; FIG. 16B). MaleapoE^(−/−)HSP27^(o/e) had a 21% reduction in en face aortic lesion area(p≦0.05; FIG. 15B) and a 24% reduction in aortic sinus lesion areacompared to their apoE^(−/−) counterparts (p≦0.05; FIG. 16B). Theseresults demonstrate that after long-term fat feeding, HSP27 maintainspersistent atheroprotection in female mice, and offers a delayed butsignificant degree of protection in male mice.

HSP27 Over-expression and Foam Cell Content: The content of theatherosclerotic lesions in both apoE^(−/−)HSP27^(o/e) and apoE^(−/−)mice was examined by histopathology. Macrophages were stained withanti-mac2 antibodies (FIG. 16A) and foam cell content was determined asa percentage of total lesion area. Female apoE^(−/−) HSP27^(o/e) had a62% decrease in foam cell content within the lesions compared to apoE−/−females (p≦0.05; FIG. 16B). Male apoE^(−/−)HSP27^(o/e) mice had a 47%decrease in foam cell area compared to apoE^(−/−) males (p≦0.05; FIG.16B). These data demonstrate that HSP27 is capable of reducing foam cellincorporation into atherosclerotic lesion areas in both male and femalemice.

Serum HSP27 Levels in Male and Female Mice: Serum levels of HSP27 wereassessed in all mice before the commencement of high fat diet, as wellas following 12 weeks of high fat diet. Prior to fat feeding, both maleand female mice had relatively low levels of HSP27 in the serum (74±23pg/ml and 211±116 pg/ml, respectively). Following 12 weeks of high-fatdiet, serum levels of HSP27 in female mice were dramatically increased(−3473±1340 pg/ml) and males had a more modest increase in serum HSP27(806±351 pg/ml) (FIG. 17). This appears to indicate that not only doesestrogen increase HSP27 release into the serum, but atherogenic stimulialso seem to cause HSP27 to be secreted over time.

In the current study, after 12 weeks of high-fat feeding, HSP27over-expression was capable of maintaining its protective effects infemale mice, with a reduction in overall lesion area as well as foamcell content. However, unlike what was observed previously in the 4-weekstudy [43], after 12 weeks of high fat diet male mice were afforded a20-25% reduction in overall lesion area, and a 48% reduction in foamcell content in these lesions. These results appear to suggest thatHSP27 over-expression is dependent upon estrogens in the early stages oflesion development, possibly through its increased secretion in responseto estrogen. However, at more advanced stages of lesion development,HSP27 seems to cause an overall reduction in foam cell content in bothmales and females, possibly through its effects in macrophage migration,adhesion, and atherogenic lipid uptake. HSP27 levels in the serum peakvery early in female mice, with 15-fold higher levels observed infemales compared to males after 4 weeks. However, after 12 weeks ofhigh-fat feeding, male HSP27 serum levels begin to increase, possibly inresponse to atherogenic lipids—an effect that was previously observed invitro. Serum levels of HSP27 in the female mice however remain high, butdo not increase relative to their levels after 4 weeks. This suggeststhat HSP27 in the serum peaks early in female mice, potentially offeringa protective effect early in lesion development. In male mice (i.e. inthe absence of estrogen) atherogenic lipids cause a slower release ofHSP27, taking longer to exert their beneficial effects.

It had previously noted that macrophages from apoE^(−/−)HSP27^(o/e) miceshow both reduced migration and adhesion compared to apoE^(−/−)macrophages. This may be another mechanism by which HSP27 is exertingits protective effects, and why reduced foam cell content in both maleand female over-expressing mice is seen.

Taken together, these data appear to suggest that HSP27 isatheroprotective in both sexes, possibly offering its beneficial effectsearlier in lesion development in female mice. In males, possibly due tolack of estrogen, HSP27 provides atheroprotection later in lesiondevelopment. Both male and female HSP27 over-expressing mice had reducedfoam cell content in their lesions compared to their non-overexpressinglittermates, confirming the role for HSP27 in the reduction in foam cellformation.

Example 10 Response to Injury

As illustrated in FIG. 18, HSP27 over-expressing (HSP27o/e) andnon-overexpressing (WT) mice were subjected to femoral artery wireinjury. The arteries were harvested 28 days post-injury, and the intimato media ratio (I/M ratio) was determined. HSP27 over-expression resultsin a decrease in neo-intima formation in both male and female mice, witha more dramatic effect observed in females.

SUMMARY

As disclosed herein, there is a reduction in circulating levels of HSP27in the serum of individuals suffering from coronary artery diseasecompared to healthy controls. This data agrees with that reported forpatients with carotid stenosis >50%, who had a 20-fold decrease incirculating HSP27 levels compared to healthy individuals¹³. Moreover,the reduction in circulating HSP27 is reversed when patients aresuffering from an acute coronary event, implying that HSP27 may besecreted into the extracellular space in response to cardiac ischemia.Previous reports in ACS patients demonstrate that within 24 hours of anacute event, HSP27 levels are increased approximately 2-fold compared toreference subjects¹⁴. Surprisingly, patients who have stable diseasehave significantly lower levels of HSP27 in the blood, which mayindicate that this protein offers some long-term protection from thedevelopment of atherosclerosis. It has been established that HSP27expression in the vessel wall is lost as atherosclerosis progresses;thus, high levels of HSP27 in both the vasculature and circulation islikely atheroprotective.

Transgenic mice studies indicate the possible in vivo ability of HSP27to protect against the development of atherosclerosis. When transgenicmice over-expressing human HSP27 were crossed with atherosclerosis-proneapoE-null mice, the female HSP27 over-expressing mice had a 41%reduction in atherosclerotic lesion area—an effect that was absent inmales. As previously shown, HSP27 is an estrogen receptor beta (ERβ)associated protein that is capable of modulating estrogen signaling viatranscriptional repression of the receptor. The current observation thatthe protective effects of HSP27 may be sex-specific highlight theoverall importance of this protein in estrogen biology. It also notedthat HSP27 in the serum is significantly higher in these same femalemice compared to their male littermates, indicating that the circulatingHSP27 is likely offering some protection against the development ofdisease, and these levels in male mice are likely not sufficientlyelevated to achieve this protective effect. The degree of correlationbetween circulating HSP27 levels and total aortic lesion area isnoteworthy, showing a correlation coefficient of 0.90. This appears toindicate that HSP27 levels in humans may be highly predictive ofatherosclerotic lesion burden.

HSP27 over-expression has been shown to be protective against a varietyof stressful and/or pathogenic stimuli (e.g. ischemia/reperfusioninjury, gastromucosal injury, nerve injury)^(17, 24-27), but these arethe first observations to date that this protection may be sex-specific.The mechanism of release of HSP27 into the serum and how ovarianhormones are affecting this process may be indicative of the enhancementof the overall protective effects of HSP27.

In vivo data of how HSP27 exits the cell and enters the serum (i.e.,extracellular space) show that HSP27 is only detectable in the serumfollowing a high-fat/atherogenic diet, in both males and females.Corresponding in vitro studies showed that by treating macrophages withhigh levels of acLDL and demonstrate that indeed HSP27 appears to bereleased into the extracellular space under these conditions. As knownin the art, oxidized LDL is capable of inducing HSP70 secretion frommacrophages²⁸. Given that HSP27 is primarily described as anintracellular protein, the mechanism by which HSP27 may exit the cellhas never been described. The fact that HSP27 does not contain signalsequences or peptides that would sort it to a traditional secretoryvesicle renders the mechanism of HSP27 to be surprising. On examiningthe lysosomal pathway, it was noted that in mouse macrophages treatedwith high levels of acLDL, HSP27 was seen in secretory-like granules,and co-localizes with the lysosome. Others have described the samemechanism for the secretion of other heat shock proteins, namelyHSP70²⁹. This mechanism for heat shock secretion is also very similar tothat described for interleukin-1, an important inflammatory stimulus³⁰.The idea that HSP27 is secreted in response to atherogenic stimuli, andthat higher levels of HSP27 correlate with lower disease burden in bothmice and humans suggests that extracellular HSP27 is perhaps an novelprotector of atherosclerotic disease and hence an attractive target forfuture therapeutics. HSP27, as a member of the heat shock proteinfamily, may also be a therapeutic target in diseases other thancardiovascular disease, such as cancer, diabetes, and the like. Inparticular, HSP27 may be a potential therapeutic target ingender-specific diseases, such as ovarian or prostate cancer, either inprophylaxis or treatment thereof. It may also serve as a diagnosticbiomarker for these and other gender- or age-dependent diseases.

A novel and important aspect of the present invention is the interactionof HSP27 and the scavenger receptor A. Not only is this interactionspecific, as demonstrated using a competitor for SR-A as well asSR-A-null macrophages, but HSP27 appears to reduce the ability of SR-Aon the surface of macrophages to engulf acLDL and acquire the foam cellphenotype. This is the first known evidence of a cell-surface receptorfor HSP27. Previous reports show that the heat shock protein family iscapable of binding a variety of receptors, namely those involved inantigen recognition and immune signaling (reviewed by³¹. For example,HSP70 has been shown to bind to and signal through the toll-likereceptors 2 and 4 (TLR-2 and -4), inducing NFκB activation and IL-6production¹⁸. Other reports show that SR-A is capable of binding Gp96 anendoplasmic-reticulum bound HSP, on antigen presenting cells³².Moreover, many studies conclude that extracellular HSPs are primarilypro-inflammatory stimuli. Recombinant HSP70 secreted from macrophages invitro induced the secretion of IL-1β, and IL-12, both pro-inflammatorycytokines.

Surprisingly, HSP27 has been shown in the present invention to haveopposite effects. When added to macrophages in vitro, HSP27 reduced theacLDL-induced secretion of IL-1β, and increased the secretion of IL-10,which imply that HSP27 primarily results in anti-inflammatory cytokineinduction. Relative to peritoneal macrophages from apoE^(−/−) mice,apoE^(−/−)HSP27^(o/e) macrophages showed decreased cell adhesion andmigration—two additional mechanisms by which vessel wall inflammationmay have been reduced in vivo with over-expression of HSP27. Takentogether, these data suggest a novel mechanism by which extracellularHSP27 is capable of preventing the uptake of atherogenic lipids andreducing inflammation associated with this uptake, therefore perhapsreducing overall atherosclerotic burden. Compounds which interact withthe SR-A receptor may also be contemplated in the context of the presentinvention, working either agonistically or antagonistically.

To further explore the mechanism by which HSP27 over-expression mayreduce the progression in a mouse-model of atherosclerosis, peritonealmacrophages were harvested from HSP27^(o/e)apoE−/− and WT apoE−/− totest the characteristics of these cells in vitro. HSP27 over-expressionresulted in a significant decrease in both cell adhesion and migration,indicating that these cells in vivo may have decreased incorporationinto atherosclerotic plaques. Combined with the observation that HSP27can reduce atherogenic lipid uptake and inflammatory cytokine release,it appears that the mechanism of atheroprotection of HSP27 involvesvirtually all hallmark processes involved in disease progression.

In the current invention, there is provided a likely novel mechanism ofHSP27 protection that involves HSP27 as an extracellular signal capableof modulating the response to atherogenic stimuli. HSP27 appears to havethe potential of preventing macrophage incorporation into the developingplaque, and subsequent ability not only to reduce foam cell formation,but also the inflammation that accompanies this process. Clearly, theimplications of this novel role for HSP27 are likely far-reaching,uncovering the possibility for HSP27 to become a new target forcholesterol-altering and anti-inflammatory therapeutics.

Other factors may be involved which contribute to the role of HSP27 inmodulating atherosclerosis. Studies, such as microarray analysis and thelike, may reveal that certain genes have similar expression profiles ofHSP27 in various states of cardiovascular disease, such asatherosclerosis. HSP27 copy number using RT-PCR may be determined. Thesestudies may also reveal additional targets or co-factors which may helpameliorate the effects of HSP27 on preventing and/or treatingatherosclerosis, or other diseases in a subject requiring prevention ortreatment thereof. Other targets and co-factors may also be contemplatedin the context of the present invention.

Hydroxy-methylglutaryl-coenzyme A reductase is the rate limiting enzymein the synthesis of cholesterol. Currently, the most popular class ofcholesterol lowering medications is characterized by the commoninhibition of 3-hydroxy-3-methylglutaryl Coenzyme A reductase, andbecause of the common terminal syllables used to name these drugs, theyare often referred to as “statins”. Treatment of patients with a“statin” is associated with altered HSP27 extracellular levels.

HSP27 may also be an important extracellular chaperone capable ofbinding key de-natured proteins and protecting the vessel wall frominsult/injury. The binding of HSP27 to serum derived proteins frompatients with and without coronary artery disease may be analyzed andquantified, particularly in real time. This may be achieved with anyknown methods in the art, such as immobilizing the HSP27 protein on achip and using surface plasmon resonance technology (Biacore Inc).

The above-described embodiments of the present invention are intended tobe examples only. Alterations, modifications and variations may beeffected to the particular embodiments by those of skill in the artwithout departing from the scope of the invention, which is definedsolely by the claims appended hereto.

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1. A method for treating cardiovascular disease in a human subject withatherosclerosis, comprising: administering to said subject atherapeutically effective amount of heat shock protein 27 or afunctional variant of heat shock protein 27 to maintain a serum level ofheat shock protein 27 or the functional variant of heat shock protein27, of at least 828 pg/mL.
 2. The method of claim 1, wherein thecardiovascular disease to be treated is atherosclerosis.
 3. The methodof claim 2, wherein the cardiovascular disease to be treated is coronaryatherosclerosis.
 4. The method of claim 1, wherein the subject isfemale.
 5. The method of claim 1, wherein the subject has coronaryartery disease.
 6. A method for treating cardiovascular disease in ahuman subject with atherosclerosis, comprising: administering to saidsubject a therapeutically effective amount of heat shock protein 27 or afunctional variant of heat shock protein 27 to increase the serum levelof heat shock protein 27 or the functional variant of heat shock protein27, to at least 828 pg/mL.
 7. The method of claim 6, wherein thecardiovascular disease to be treated is atherosclerosis.
 8. The methodof claim 6, wherein the cardiovascular disease to be treated is coronaryatherosclerosis.
 9. The method of claim 6, wherein the subject isfemale.
 10. The method of claim 6, wherein the subject has coronaryartery disease.